Natural products have historically been the source of most of the microtubule (MT)-targeting small molecules whose properties have allowed them to become useful drugs. That remains true of most but not all of the compounds in this study. Some, such as the new MT-stabilizing compound peloruside, are natural products, as is the clinically established MT-stabilizer taxol. Others, such as analogs of the MT-stabilizing epothilones, are semisynthetic derivatives based on known natural compounds. Others still are totally synthetic compounds. We have investigated new binding sites on tubulin for anti-MT drugs, as well as the results of drug binding at these, or the longer-known sites, on the properties of MT and the effects on cells. The new binding sites are for the synthetic MT destabilizer, oryzalin, and the natural product MT stabilizer, peloruside. The effects on cells involve these drugs as well as more established drugs, especially clinical agents. In order to understand the activity of the new microtubule stabilizer, peloruside, it is necessary to know the details of the binding site. We have already shown by mass spectrometric studies and molecular modeling that this compound binds to a site on beta tubulin quite distinct from that of taxol, a clinically important MT-stabilizing drug. Selecting and mapping mutations in human tubulin that confer resistance to peloruside have confirmed our mass spectrometry studies, and allowed an improved understanding of the binding site, how occupancy alters MT stability, and how this differs from taxol action. We have now defined this binding site at high resolution by mapping the location of several independent mutations that confer resistance to the cytotoxic action of peloruside. These results confirm the lower-resolution results we previously obtained using mass spectrometry, but provide significant new details. The detailed knowledge of the binding site reinforces the independence of this binding site from that for taxol, despite the similarity of their actions. This knowledge also strongly suggests a detailed molecular mechanism different from that of taxol, but which stabilizes the microtubule structure just as taxol does. Since these two mechanisms are different in detail, this may inform a rational combination of these drugs to maximize the synergistic action of the two together which has already been demonstrated. We hope to use this knowledge to understand the differing mechanisms of peloruside and taxol, and provide a basis for combination of these drugs clinically. It is already clear from the binding site mapping and from preclinical studies that taxol and peloruside stabilize MT by different mechanisms. Structural study of the two binding sites suggests a differing balance of longitudinal and lateral stabilization in the MT polymer, suggesting that the mechanical properties of the MT may differ with the two drugs. Unperturbed MT are the most rigid intracellular protein polymers known, and taxol increases their flexibility 10-fold. We are measuring the rigidity of individual fluorescent MT after binding of taxol or peloruside in order to relate differences in binding site structures to differences in MT properties. This understanding could provide an explanation for the synergistic effect observed for combinations of these drugs in preclinical cellular models. The roles of MT extend throughout the life of the cell, not only in mitosis, but also in the 98+% of the cell cycle that is not mitosis. These vital roles include those from above establishing cellular polarity, supporting intracellular transport and signaling, and allowing directionality in cell movements. MT-targeting drugs are active in all cells, not only in mitotic ones, and indeed some targets of clinical use of anti-MT drugs are post-mitotic cells. We have argued that even in clinical settings where intuition says that mitosis is the target, such as in patient tumors, data indicate that MT-targeting drugs are effective due to interference with non-mitotic processes, such as those mentioned above. We plan to combine the experimental approaches described to obtain a better understanding of the non-mitotic processes that are targeted by the action of anti-MT drugs in order to improve the clinical usefulness of these agents. An attempt to improve on the clinical effectiveness of anti-microtubule drugs was based on the assumption that these agents act via inhibition of mitosis, while their neurological side effects were unrelated to mitosis. Based on this view, a number of compounds have been developed by multiple laboratories and pharmaceutical companies that target proteins that are only expressed during mitosis. These mitosis-specific drugs were developed, at great expense, and put in clinical trials where they have shown very little activity against patient tumors. We combined results of all of the clinical trials and compared them to understand why they failed. We concluded that the problem is that mitosis is too rare in tumors for these drugs to be clinically effective, and that therefore the effectiveness seen with anti-microtubule agents is due to activity against some non-mitotic process(es) that are microtubule-dependent. We are actively seeking what these process(es) might be.